Generation And Identification Of Monoclonal Antibody For Anti-ractopamine And Establishing Immunoassay Method Of Ractopamine

Ractopamine(RCT), one of the synthesizedβ2-adrenergic agonists, used for asthma and other human respiratory system disease in the early time. It has been approved that ractopamine has a powerful effect promoting animal growth. It can increases accretion of skeletal muscle mass and decrease accretion of body fat. For this purpose, RCT has been illegally used as a growth-promoting agent to improve feed conversion efficiency and enhance lean meat to fat ration. As a kinds of growth-promoting agent the dosage of ractopamine far more exceeds the normal curing dosage, the accumulative residue of ractopamine in food producing animals can cause poisoning symptoms such as dizzy, quickening heartbeat, muscle dindling, palpitation and so on in human. The government has proclaimedβ2-adrenergic agonists is prohibited as additive of animals feed. FDA and WHO proposed maximal residue of ractopamine in urine did not exceed 1.2ng/ml, 0.6ng/g in tissue. The government of HONGKONG has legislated prohibitingβ2-adrenergic agonists such as ractopamine as feed additive. As a member of the WTO the residue ofβ2-adrenergic agonists such as ractopamine will badly impacts the export of animal products of our country. It is very important how to establish a kind of credible, sensitive and quick assaying method.Ractopamine conjugated antigen (RCT-KLH and RCT-OVA) were procuced by coupling ractopamine with Keyhole limpet haemocyanin (KLH) or Ovalbumin (OVA) through glutarate anhydride and isobutychloroformate. RCT-KLH was used as immune antigen RCT-OVA as coating antigen. Anti-RCT monoclonal antibodies were produced by conventional cell fusion technology. The spleen cells of immunized Blab/c mouses were fused with SP2/0 plasmacytoma cells using PEG4000, and selected with HAT and HT medium. Three hybridoma cell lines of 2B6,3D3,2F7 were screened for specificity to RCT with RCT-OVA using an indirect ELISA, and cloned by dilution method for three times, which secrete high affinity monoclonal antibodies anti-RCT with indirect ELISA titers of 1:8.0×104,1:3.2×105,1:4.0×104.On the basis of prohibitive ELISA, Anti-RCT monoclonal antibodies showed good sensitivity with an IC50 of 40.5ng/ml toward RCT, more than 3906ng/ml towards CL, Norepinephrine, Epinephine, Isoprenaline .With Anti-RCT monoclonal antibodies we established method immunoassay of RCT based on prohibitive ELISA. We also optimized the condition so as to provide gist for immunoassay kit.